Non-toxic solvent for chromogenic substrate solution and uses thereof

ABSTRACT

The present invention relates to a non-toxic dipolar solvent for chromogenic substrate for detecting presence of lacZ gene and/or gene activity, which comprises a stabilizing amount of a solubilizing agent. The present invention also relates to a method for inducing lac operon in screening assay, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce the lac operon. The present invention further relates to a method for detecting the presence of bacteria, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce detection of the bacteria.

PRIORITY

This application claims priority to co-pending U.S. application Ser. No. 10/533,544, which claims priority to PCT/CA 03/01690 filed Oct. 31, 2003, which claims priority to U.S. application Ser. No. 60/422,873 filed Nov. 1, 2002 each of which is expressly incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

This invention relates to a non-toxic solvent for preparing chromogenic substrate solution and uses thereof.

(b) Description of Prior Art

Many of the cloning and expression vectors in current use (e.g. the pUC series) carry a short segment of E. coli DNA that contains the regulatory sequences and the coding information for the first 146 amino acids of the β-galactosidase gene (lacZ). Embedded in this coding region is a polycloning site that does not disrupt the reading frame but results in the harmless interpolation of a small number of amino acids into the amino-terminal fragment of β-galactosidase. Vectors of this type are used in host cells that code for the carboxy-terminal portion of β-galactosidase. Although neither the host-encoded nor the plasmid-encoded fragments are themselves active, they can associate to form an enzymatically active protein. This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by β-galactosidase-negative mutants that have the operator-proximal region intact, is called α-complementation. The Lac+ bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate 5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal) (Horwitz et al. 1964. Substrates for cytochemical demonstration of enzyme activity. I. Some substituted 3-indoxyl-β-D-galactopyranosides. J. Med. Chem. 7:574.). However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in the production of an amino-terminal fragment that is not capable of α-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple color test has greatly simplified the identification of recombinants constructed in plasmid vectors of this type. It is easily possible to screen many thousands of colonies visually and to recognize colonies that carry putative recombinant plasmids. The structure of these plasmids is then verified by restriction analysis of mini-preparations of plasmid DNA.

To a pre-made LB agar plate containing the appropriate antibiotics, a quantity of a stock solution of X-gal (20 mg/ml in dimethylformamide (DMF) or Dimethyl sulfoxide (DMSO)) and a quantity of a solution of isopropylthio-β-D-galactoside (IPTG) is added. The stock solution of X-gal is usually prepared by dissolving X-gal in dimethylformamide or Dimethyl sulfoxide which is a toxic solvent presenting also the drawback of providing solutions that are not stable through time.

IPTG is an important addition to the blue-white screening. The vectors carrying a segment of DNA derived from the lac operon of E. coli that codes for the amino-terminal fragment of β-galactosidase can be induced by isopropylthio-β-D-galactoside (IPTG). Bacteria exposed to the gratuitous inducer IPTG synthesize both fragments of the enzyme and form blue colonies when plated on media containing the chromogenic substrate 5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal).

It would be highly desirable to be provided with new solvents that are non toxic for preparing chromogenic substrate solutions used in screening assays, these solvents providing an extended stability of the chromogenic substrate solution.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a non-toxic dipolar solvent for chromogenic substrate for detecting presence of lacZ gene and/or lacZ gene activity, which comprises a stabilizing amount of a solubilizing agent.

The solvent in accordance with a preferred embodiment of the present invention, wherein the solvent is a microemulsion.

The solvent in accordance with a preferred embodiment of the present invention, wherein the solubilizing agent is at least one selected from the group consisting of 1-Methylpyrrolidone (NMP), N′-dimethyl propylene urea (DMPU), Propylene carbonate (PC) and essential oil.

The solvent in accordance with a preferred embodiment of the present invention, wherein the essential oil is present in an effective solubilizing concentration for dissolving the chromogenic substrate.

The solvent in accordance with a preferred embodiment of the present invention, wherein the essential oil is selected from the group consisting of Abies alba, Aniba roseodora, Cedrus atlantica, Citrus aurantifolia, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus paradisi, Citrus reticulata, Citrus sinensis, Cupressus sempervirens, Juniperus communis, Juniperus virginiana, Picea mariana, Pinus sylvestris, Ravensara aromatica, Rosmarinus officinalis, citrus extracts, pine terpenoids, conifers extracts, limonene oil and linseed oil.

In accordance with the present invention, there is provided a composition for detecting the presence of lacZ gene and/or lacZ gene activity comprising the solvent of the present invention and an effective amount of chromogenic substrate.

In accordance with the present invention, there is provided a method for inducing lac operon in screening assay, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce the lac operon.

The method in accordance with a preferred embodiment of the present invention, the lac operon being induced in one selected from the group consisting of E. Coli, Bacillus subtilis, phage, or in situ tissues.

In accordance with the present invention, there is provided a method for detecting the presence of bacteria, comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce detection of the bacteria.

For the purpose of the present invention the following terms are defined below.

The term “chromogenic substrate” is intended to mean a substrate that produces a color when contacted with an appropriate reagent. The chromogenic substrate can be one of, but not limited to, X-Gal and IPTG.

All references herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a bacterial culture exhibiting a strong blue color indicative of lac operon induction without the presence of IPTG when X-gal dissolved in essential oils;

FIG. 2 illustrates the results of plating a ligation/transformation onto LB plates containing X-gal dissolved in dimethylformamide (DMF);

FIG. 3 illustrates the results of plating a ligation/transformation onto LB plates containing X-gal dissolved in NMP and methanol;

FIG. 4 illustrates the results of plating a ligation/transformation onto LB plates containing X-gal dissolved in NMP, sea pine turpentine and methanol; and

FIG. 5 illustrates the results of plating a ligation/transformation onto LB plates containing X-gal solution aged of 16 months;

FIG. 6 illustrates the aging of LB plates at 4° C., room temperature, 37° C. and 65° C. (in the clockwise direction); and

FIGS. 7A-7C illustrate LB plates after 2 weeks at various temperatures.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided non-toxic solvents for dissolving and stabilizing enzyme substrate used in screening assays.

One enzyme substrate widely used is X-gal, which is a dipolar molecule having the formula I:

-   5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside

The X-gal solutions prepared with a non-toxic dipolar solvent of the present invention, for example 1-Methylpyrrolidone (NMP), N′-dimethyl propylene urea (DMPU), Propylene carbonate (PC), essential oils or a combination of these, are very stable. In solution at 4° C., the X-gal will keep its activity for more than 6 months. If used to be poured in agar plates containing the proper antibiotic, these plates will remain active and usable for at least 3 months.

As an example of its non-toxicity, NMP is known as rapidly absorbed and eliminated. It is currently used intravenously in horses as a preanaesthetic. It is also used as an excipient in topical pharmaceutical formulations in human medicine and in cosmetics.

Essential oil mixes can be used to create a dipolar environment allowing X-gal dissolution. For instance, a combination of citrus extracts, pine terpenoids, limonene and linseed oil was shown to create an environment allowing the proper dissolution of X-gal.

Non-toxic micro emulsions can also be used to dissolve X-gal. A micro emulsion is a thermodynamically stable dispersion of one liquid phase into another, stabilized by an interfacial film of surfactant. This dispersion may be either oil-in-water or water-in-oil. Micro emulsions are typically clear solutions, as the droplet diameter is approximately 100 nanometers or less. The interfacial tension between the two phases is extremely low. Emulsions are in contrast unstable, the suspended droplets will eventually agglomerate and the dispersed phase will phase separate. Emulsion droplet sizes are much larger, typically one micron or more, resulting in a cloudy or milky dispersion. The nature of an emulsion may depend on the mixing of the ingredients and the amount of energy put into the mixing process.

A combination of essential oil was extracted with chloroform in order to remove part of the oil phase, and create a semi-precipitated emulsion. For example, 20 ml of citrus extract, pine terpenoids, limonene oils and linseed oil was mixed with 20 ml of Chloroform by vigorously shaking in a 50 ml falcon tube. The resulting mix was allowed to let stand for approximately one day, and then the upper phase was transferred to a 15 ml falcon tube. The obtained phase is a milky emulsion. It dissolves X-gal very well. If we let stand the milky phase for approximately one week, it will show the development of 5 distinct phases. Isolating each phase, it was interesting to observe that only phase #2, the milky sub-phase, then still an emulsion, can be used as an active solution to dissolve X-gal. The active phase is yellow milky cloudy in appearance. Using 500 μl completely dissolve 0.01 g of X-gal; however, it gives a very opaque and viscous solution in appearance.

Micro emulsions are proper to create dipolar environment allowing a complete dissolution of X-gal. A mixture of oil, egg yolk or lecithine and acetic acid was sufficient to dissolve X-gal and show blue bacteria when used for its function as spread on an agar surface. Linseed oil, which has an average composition of different fatty acids (C16:0 palmitic acid 4-9%, C18:0 stearic acid 2-4%, C18:1 oleic acid 14-39%, C18:2 linoleic acid 7-19%, C18:3 linolenic acid 35-66%) is a known carrier for lipophilic molecules (as essential oils) and can act as a compound of the oily phase of the emulsion.

A micro emulsion is ideally made of a non-polar liquid mixed with a polar liquid in the presence of a surfactant or amphiphile, which is ideally a molecule carrying both polar and non-polar charges. In our situation, the surfactant, or amphiphilic molecule is X-gal. The role of the surfactant is to reduce the interfacial tension between two partially miscible or immiscible fluids below that obtained when no surfactant is present.

As shown in Table 1, variation in the composition of the micro-emulsion has an impact on the temperature flash point, which is a concern for transportation purposes.

TABLE 1 X-Gal with Flash point (° C.) 10% water/90% NMP >93 5% water/95% NMP 88 1% seapine/10% water/89% NMP 76 1% seapine/99% NMP 73 10% seapine/90% NMp 47.5

As well, IPTG is a usual and relatively essential addition to the cloning process. Omitting IPTG from the growth medium will decrease the expression level from plac, blue/white selection is usually not possible in the absence of IPTG. E. coli lac operon consists of a promoter, a transcriptional regulatory site called the operator (O), a CAP binding site (c), and three structural genes (lacZ, lacY and lacA) that are transcribed as a single polycistronic mRNA. Transcription of the lac operon is regulated by the lac repressor protein (lad) which is encoded on a gene physically linked to the lac operon. lac operon inducers, such as IPTG, inactivate the lac repressor protein resulting in transcriptional de-repression of the lac operon. It is possible to artificially induce the lac operon using a nonmetabolizable allolactose analogue, isopropylthiogalactoside (IPTG), which binds to the lac repressor protein.

It is shown here that essential oils are replacing IPTG to induce the lac operon. It was found that compositions comprising essential oils not only dissolves X-gal, but also enhances the blue color without the need of IPTG. Among different tested essential oils, the most interesting alternative to IPTG is a small amount of the essential oil Sea Pine. Other essential oils extracted from spruce, pine or other conifers are also candidate to replace IPTG. Using such oil instead of IPTG represents different benefits, being non-toxic, all natural, easily biodegradable, low cost and most importantly, already as a liquid solution, ready to use.

Monoterpenes and sesquiterpenes were shown to be successful in dissolving X-gal and replacing IPTG in screening clones using the blue/white X-gal selection method.

In addition to X-gal, it is also possible to dissolve IPTG with the same solutions for cloning with bacterial systems needing that stimulation (see Table 2 for examples). These same solutions allow stable dissolution of Ampicillin, tetracycline and Chloramphenicol, providing an approach wherein a complete solution is available to the user. To dissolve Kanamycin, a solution with a base of micelles in aqueous solution is essential.

TABLE 2 Strain (lacZ)M15 lacIq F′ Cam R Kan R Str R Tet R Comments DH5-alpha x Invitrogen: (lacZYA)U169 DH5-alpha E x Invitrogen; (lacZYA)U169 DH5-alpha F′ x x Invitrogen; (lacZYA)U169 DH5-alpha x Invitrogen; T1R (lacZYA)U169 DH10B x Invitrogen DH10Bac x Invitrogen; for producing recombinant baculovirus molecules DH10B T1 R x Invitrogen GeneHogs x Invitrogen; D10B derivative INV-alpha F′ x x Invitrogen, (lacZYA)U169 JM83 x x ATCC 35607 Select 96 x Promega TB1 x x NEB; comes with pMAL system TOP10 x Invitrogen TOP10/P3 x x x x Invitrogen; AMP R ABLE C x x x x x Stratagene; lower plasmid copy number ABLE K x x x x x stratagene; lower plasmid copy number AD494 BB4 x x x x =LE392.23 BMH 71-18 x x x x mutS DH5-alpha F′ x x x x Invitrogen IQ DH-alpha FT x x x x Invitrogen; (lacZYA)U169 DH11S x x x Invitrogen; mainly for ssDNA production with phagemid/m13 DH12S x x x x Invitrogen Electro Ten x x x x x Stratagene blue ER1727 x x x x x NEB ER2267 x x x NEB ER2738 x x x NEB INV110 x x x x Invitrogen; to produce umethylated DNA JM101 x x x ATCC33876 JM103 x x x x ATCC 39403 JM105 x x x x ATCC47016 JM107 x x x ATCC47014 JM109 x x x ATCC 53323; to maintain F′ grow on M9 with 1 mM thiamine JM110 x x x x ATCC 47013 NM522 x x x ATCC 47000 NM527 SCS110 x x x x Stratagene Solo Pack x x x x x Stratagene gold SOLR x x x x Stratagene; excision assist strain Stbl4 x x x x Invitrogen; to clone unstable DNA SURE x x x x x Stratagene SURE-2 x x x x x x Stratagene TG1 x x x TKX1 x x x x x Stratagene; for prot production TOP10 F′ x x x x x W3110 XL1-Blue x x x x Stratagene XL1-Blue x x x x Stratagene MRF′ XL1-Blue x x x x x Stratagene MRF′ Kan XL2-Blue x x x x x Stratagene XL2-Blue x x x x x Stratagene MRF′ XL10gold x x x x x Stratagene XL10gold x x x x x Stratagene Kan R XL mutS Kan S x x x x Stratagene XL mutS Kan R x x x x x stratagene XLOLR x x x x Stratagene; excision assist strain XPORT x x x G1698 x C1724 x 96 pack gold x x x x x EC100 x Epicentre; pIndigo EC300 x Epicentre; copy control BAC CC300 x Epicentre; copy control BAC EPI100 ? ? Epicentre; fosmid EPI300 ? ? Epicentre; fosmid AG1 DH1 derivative BL21 (DE3) x for prot expression; some also Cam R or Kan R BNN93 C600 ATCC 23724 CJ236 x x DB3.1 Invitrogen; for propagating vector with ccdB gene DH1 ATCC 33849 DH5 alpha Invitrogen; MCR (lacZYA)U169

In a preferred embodiment of the present invention, individual LB plates are prepared adding 100 μl of X-gal solution to the surface of a LB plate being at room temperature and spreading evenly across the surface. The plate is dried before use. X-gal containing LB+Amp plates are stable for up to 90 days when stored at 4° C.

Batches can be prepared by aseptically adding X-gal solution directly to melted LB agar (temperature 50° C.). 100 μl of the solution should be used for every Petri dish. For example, for each 500 ml add 2000 μl of X-gal solution. Mix well (for 3 to 5 minutes) and pour as you normally would. Let cool. X-gal LB plates are stable for at least 3 months when stored at 4° C. X-gal can be added before or after the addition of selective antibiotics to the medium. For best mixing results, a magnetic stir bar should be added during the autoclaving process or a sterile magnetic stir bar could be ascetically added after autoclaving.

In a preferred embodiment of the present invention, X-gal solution is prepared as follow:

NMP or DMPU Only

Add 7 ml of either NMP or DMPU to 1 gram of X-gal. Stir until dissolved and bring to 10 ml with NMP or DMPU (whichever is already used).

NMP or DMPU with Essential Oil

Add 3.5 ml of either NMP or DMPU to 1 gram of X-gal. Stir until dissolved and bring to 5 ml with NMP or DMPU (whichever is already used). Mix with 5 ml of essential oil until thoroughly mixed.

NMP/Methanol Solution

Add 7 ml of either NMP or DMPU to 1 gram of X-gal and stir until dissolved. Bring to 10 ml with NMP or DMPU (whichever is already used). Add 90 ml of methanol and mix thoroughly.

Example 1 Essential Oils as Solvent and IPTG Replacement

Using 0.01 g X-gal in 500 μl of TURPENOID NATURAL® (comprising a combination of citrus extracts, pine terpenoids, limonene and linseed oil) provides excellent dissolution of X-gal and without affecting bacterial growth. It also provides a strong blue color without the need of IPTG, as shown in FIG. 1.

Example 2 Dissolution of X-gal

10 mg of X-gal powder were successfully dissolved in the solvents described in the Table 3 below.

TABLE 3 Mix T1− Citrus lemon/Citrus 700 μl sinensis Pinus pinaster 300 μl Mix T2− Cupressus sempervirens  50 μl Pinene (Sigma)  50 μl Campher oil 100 μl Citrus sinensis 800 μl Mix T3− NMP 100 μl Methanol (only to 900 μl increase total volume): % NMP: 10% Mix T4− PC 200 μl Methanol (only to 400 μl increase total volume): % PC: 33%

Example 3 Dissolution in NPM and NMP/Methanol

A 10× X-gal/NMP solution could be stored at ⁻20° C. without freezing. Over time, the solution will take a very pale yellow color that does not seem to darken over time. A 10× X-gal/NMP solution is easily diluted to 1× in anhydrous methanol or 95% ethanol. When diluted in methanol or ethanol, the solution will not freeze.

When the 1× X-gal/NMP/methanol is stored for 16 weeks at 4° C., there is no loss in X-gal activity as measured by applying 100 μl to a LB plate, streaking E. coli pUC19 transfectant, incubation at 37° C. and examination for blue colonies after 16 hours.

Tables 4 and 5 are showing the results obtained by testing the activity of X-gal in solution with different solvent on a weekly basis. In Table 4, the tested solutions were stored at 4° C. At weekly intervals, 100 μl was applied to an LB+Amp plate and streaked with E. coli containing pUC19. The plate was incubated overnight at 37° C. The following morning the plate was examined for blue colonies. In Table 5, LB+Amp plates were prepared and stored at 4° C. At weekly intervals, a plate was removed and streaked with E. coli containing pUC19. The plate was incubated at 37° C. overnight. The blueness of the colonies was then scored.

TABLE 4 Weekly Testing of X-gal/NMP/Methanol solution Week Test Results 0 + 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 + 16 + 17 + 18 + 19 + 20 +

TABLE 5 Weekly testing of X-gal/NMP or X-gal/NMP/oil Date NMP plate Oil + NMP plate 0 + + 1 + + 2 + + 3 + + 4 + + 5 + + 6 + + 7 + + 8 + + 9 + + 10 + + 11 + + 12 + + 13 + + 14 + +

Example 4 Dissolution with Essential Oils

It was shown that when X-gal was first dissolved in NMP, mixed one to one with essential oil, and then diluted in methanol to 10 mg/ml the mixture would ultimately yield colonies that are darker blue than when X-gal is made up in dimethylformamide.

This work was repeated and expanded to include other essential oils. The oils tested were: Natural Orange Terpene Solvent (Eco-House); Blue Gum Eucalyptus Organic (Divine Essense); Atlas cedarwood (Pranarom); Sea Pine Turpentine (Pranarom) and Natural Turpenoid.

In this experiment, a 100 mg/ml solution of X-gal in NMP was prepared. A one to one mix with each essential oil was then made using this solution. The net X-gal concentration is now 50 mg/ml. This solution was then dilute 5× with 100% methanol such that the final concentration of X-gal is 10 mg/ml. One hundred microliters of this solution is then applied per LB plate.

As a control, 50 μl of a 20 mg/ml solution of X-gal dissolved in dimethylformamide was applied to one LB plate.

E. coli containing pUC19 was streaked onto each “X-gal spread” LB plate for isolated colonies. The plates were incubated at 37° C. overnight. After incubation, the growth on each X-gal plate was scored for blueness. The ranking was as follows (least blue to most blue): A (least); B and X-gal/DMF; C, D and E (most blue).

The results showed that when either Atlas Cedarwood, Sea Pine Turpenine or Natural Turpenoid were mixed with X-gal/NMP/methanol there was an enhanced blueness of the E. Coli pUC containing bacteria.

During assays with Sea Pine Turpentine, the amount of Sea Pine Turpentine added to the X-gal/NMP solution was serially diluted one in two four times, mixed with methanol and then spread onto LB plates. The amount of X-gal added to each plate was the same. The plates were then streaked with E coli containing pUC19 and incubated overnight.

An examination of the plates showed that the bluest colonies were those obtained with the original amount of Sea Pine Turpentine gave the deepest blue color.

In the previous experiments, the final X-gal concentration was 10 mg/ml. The concentration of X-gal was reduced to 7.5, 5 and 2.5 mg/ml. Plates were prepared and streaked. The net result was that there was a significant drop in blue coloration when the X-gal was dropped from 7.5 to 2.5 mg/ml. In a preferred embodiment of the present invention, X-gal is in a concentration of 10 mg/ml.

Ligation assays were performed using lambda DNA digested with PstI and pUC19 digested with PstI and CTAP treated. The completed ligation was transformed into DH5α and plated onto LB plates containing:

(a) X-gal dissolved in Dimethylformamide (b) X-gal dissolved in NMP and methanol (c) X-gal dissolved in NMP, Sea Pine Turpentine and methanol

The plates were incubated overnight. The results for a, b and c are shown in FIGS. 2, 3 and 4, respectively. For all three plating, there were both white and blue colonies. Moreover, it is shown that the blues colonies of FIG. 4 are of a more intense blue than the ones of FIGS. 2 and 3.

It is possible to incorporate X-gal directly in molten LB agar. To test this with the X-gal solution of the present invention, 500 ml of LB agar was made, autoclaved and cooled to about 50° C. To this 2 ml of a 10 mg/ml X-gal/NMP/SeaPine/methanol solution was added and mixed. Upon the addition of the X-gal solution, there was a cloudy appearance throughout the agar as it mixed. Mixing for 3 to 4 minutes did not disperse the cloudiness. However, when the plates were poured, the cloudiness dispersed upon cooling and solidification. A slight surface cloudiness was noted several hours later when the plates were inverted for incubation overnight at room temperature. However, the next day the plates looked normal.

One plate was used to streak a white and a blue colony and incubated overnight at 37° C.

Two plates were left at 25° C. These two plates served as shipping simulators. The plates were tested after 5 days and were streaked with a blue and white colony and incubated overnight.

Aging studies have been set up for the liquid product both with and without Sea Pine Turpentine. The studies conducted with the X-gal/NMP/methanol solution shown that the product is stable for 17 weeks at 4° C. with only a minor pale yellow color developing over time. This has being conducted at the same time with the X-gal/NMP/SeaPine/methanol product.

Example 5 Aging Studies

X-gal solution using NMP and/or essential oil were shown to have an improve shelf-life. Table 6 is providing life span of X-gal solutions.

TABLE 6 Temperature Time  4° C. 16 months Room temperature  4 months 37° C. 1 month 65° C. 1 month

Moreover, it had been found that XGaI Petri dishes were still active when conserved at 4° C. during as long as 12 months.

Method and Results

Longest-term recipe is 1 Jun. 2002 in amber bottles and stored at 4° C. The recipe is 10% NMP and 90% methanol. The solution was last test Sep. 25, 2003 and found to be functioning fine (see FIG. 5). Currently, only a pale yellow color and not crystals.

X-gal containing LB plates were prepared Jul. 30, 2002. Two sets of plates were poured and stored at 4° C. only. The first set was X-gal dissolved in NMP then an equal volume of natural turpenoid extract was added followed by ethanol. The final X-gal concentration in the solution was 10 mg/ml. Ampicillin prepared in water was added separately to the molten agar before pouring.

Each week one plate was removed, streaked with pUC/DH5α and DH5α and incubated at 37° C. overnight. During the course of the experiment, the streaked pUC/DH5α always turned blue and the DH5α did not grow. Up until Jul. 30, 2003, both Ampicillin and X-gal were functioning.

Tables 7-9 provide results from more aging studies performed with solutions in accordance with the present invention

TABLE 7 10% NMP Main Ingredients May 12 June 3 June 9 June 13 June 27 July 8 Aug 5 Aug 15 Sept 22 X-gal (ethanol) No crystals, Clear, no Clear, no Yellow, no Yellow, no Yellow, no Yellow Slightly Slightly May 5, 2003 clear crystals crystals crystals crystals color, no crystals, yellow, no yellow, no solution, ok ok crystals ok crystals ok crystals ok Ok X-gal No crystals, Clear, no Clear, no Clear, no Clear, no Clear, no Clear, no Clear, no Clear, no (methanol) clear crystals crystals ok crystals ok crystals ok crystals crystals crystals crystals May 1, 2003 solution ok ok Ok Ampicillin + No crystals, Yellow, yellow yellow color, yellow, no Yellow, no Yellow, no Yellow, no Yellow, no X-gal slight yellow No crystals, color, no no crystals crystals Ok crystals crystals, crystals crystals May 6, 2003 color Ok ok crystals Ok ok ok Ok ok Ok ok Ok ok Ok ok Ok ok Amp in NMP + NA Pale yellow Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no X-gal Ok ok crystals crystals crystals crystals crystals crystals crystals Ok Ok May 15, 2003 Ok, ok Ok, ok Ok, ok Ok ok Ok ok Chloramphenic No crystals, Pale yellow, Pale yellow, Pale yellow, Pale yellow, Pale yellow, Pale yellow, Pale yellow, Pale yellow, no of IPTG (low) + clear no crystals, no crystals no crystals no crystals no crystals no crystals no crystals crystals X-gal solution Ok, ok Ok ok Ok ok Ok Ok Ok ok Ok ok Ok ok May 5, 2003 Ok, ok Chloramphenicol No crystals, Pale yellow, Pale yellow Pale yellow, yellow Pale yellow, Pale yellow, Pale yellow, Pale yellow, no IPTG (high) + clear no crystals, no crystals no crystals color, no no crystals, no crystals, no crystals crystals Ok Ok X-gal solution Ok, ok Ok ok Ok ok crystals Ok ok, ok ok ok May 5, 2003 Ok, ok ok Tetracycline* + Yellow, no Yellow, Darker Darker Darker Dark yellow, Dark yellow, Yellow, no Yellow, no X-gal crystals, clear, no yellow, no yellow, no yellow no crystals no crystals crystals, ok crystals, ok ok (Apr. 4, 2003) No growth crystals, crystals crystals color, no Ok ok Ok ok ok of DH5α No growth Ok ok crystals of DH5α ok ok Tetracycline Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no (low) + X-gal crystals crystals crystals crystals crystals crystals crystals, crystals crystals Ok Ok (May 5, 2003) Ok ok Ok, ok Tetracycline Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no Yellow, no (high) + X-gal crystals crystals crystals crystals crystals crystals crystals, crystals crystals (May 5, 2003) Ok, ok Ok, ok Ok Ok

TABLE 8 AGING TEST #1 Aging test Aging test Formulations Start date (# weeks) Result (# weeks) DMF + X-Gal RT 17 Sep. 2003 1 week Appearance of soln: clear, Intensity 4+ 4 weeks −20 mid-july-03 ~9 weeks Appearance of soln: clear, Intensity 4+ ~12 weeks BT lot no. 024 (25% NMP) 65 C.  17 Sep. 2003 1 week Appearance of soln: yellow, Intensity 4+ 4 weeks 37 C.  17 Sep. 2003 1 week Appearance of soln: light yellow, Intensity 4+ 4 weeks RT 21 Aug. 2003 5 weeks Appearance of soln: clear, Intensity 4+ 8 weeks 4 C. 5 Aug. 2003 ~7 weeeks Appearance of soln: clear, Intensity 4+ ~10 weeks BT lot no. 004 RT 21 Aug. 2003 5 weeks Appearance of soln: light yellow, Intensity 4+ 8 weeks 4 C. 18 Feb. 2003 ~31 weeks Appearance of soln: light yellow, Intensity 4+ ~34 weeks BT Amp, lot 019 RT 21 Aug. 2003 5 weeks Appearance of soln: yellow, Intensity 3+ 8 weeks 4 C. 16 May 2003 ~18 weeks Appearance of soln: yellow, Intensity 3+ ~21 weeks All were tested with DH5 + TrueBlue BT lot no. 024 (25% NMP) has an intensity rated higher than others probably because the resulting evaporation made it more [ ]

TABLE 9 AGING TEST NEW FORMULATIONS Formulations Start date Week 2 Result W4 (M1) A + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: VERYyellow, Intensity 3+ 28 Oct. 2003 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 −20 28 Sep. 2003 28 Oct. 2003 D + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: VERYyellow, Intensity 3+ 28 Oct. 2003 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ −20 28 Sep. 2003 28 Oct. 2003 K + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: brown-orange, Intensity 3+ 28 Oct. 2003 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 −20 28 Sep. 2003 28 Oct. 2003 BT PLUS 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: greenish-brown, Intensity 3+ 28 Oct. 2003 lot no. 028, 25% NMP 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 −20 28 Sep. 2003 28 Oct. 2003 DMF + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: orange, Intensity −−− 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 28 Oct. 2003 −20 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 DMSO + X-Gal/IPTG 65 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: greenish-brown, Intensity −−− 37 C. 28 Sep. 2003 14 Oct. 2003 Appearance of soln: light yellow, Intensity 4+ 28 Oct. 2003 RT 28 Sep. 2003 28 Oct. 2003  4 C. 28 Sep. 2003 28 Oct. 2003 −20 28 Sep. 2003 14 Oct. 2003 Appearance of soln: clear, Intensity 4+ 28 Oct. 2003 Test 14 oct was done with DH5 + TrueBlue Test 28 oct was done with xL1 + PUC (also solutions are 65C were rought back to 1400 uL)

In FIG. 6, dishes from the BT lot no. 024, as described in Table 8 are illustrated. The two top left dish was conserved at 4° C., the top right dish was conserved at room temperature, the bottom left dish was conserved at 37° C. and the bottom right dish was conserved at 65° C.

In FIG. 7A, dishes from the lot A, as described in Table 9, are illustrated. The top dish was conserved two weeks at 4° C., the bottom left dish was conserved two weeks at 37° C. and the bottom right dish was conserved two weeks at 65° C.

In FIG. 7B, dishes from the lot 028, as described in Table 9, are illustrated. The top dish was conserved two weeks at 4° C., the bottom left dish was conserved two weeks at 37° C. and the bottom right dish was conserved two weeks at 65° C.

In FIG. 7C, dishes from the lot DMSO, as described in Table 9, are illustrated. The top dish was conserved two weeks at −20° C., the bottom left dish was conserved two weeks at 37° C. and the bottom right dish was conserved two weeks at 65° C.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. 

1. A solvent for a chromogenic substrate for detecting presence, activity, expression, and/or non-expression of a lacZ gene, the solvent comprising at least one compound selected from the group consisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), an essential oil selected from the group consisting of Abies alba, Aniba roseodora, Cedrus atlantica, Citrus aurantifolia, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus paradisi, Citrus reticulata, Citrus sinensis, Cupressus sempervirens, Juniperus communis, Juniperus virginiana, Picea mariana, Pinus pinaster, Pinus sylvestris, Ravensara aromatica, Rosmarinus officinalis, citrus extracts, pine terpenoids, conifers extracts, limonene oil, linseed oil, and combinations thereof, and combinations thereof.
 2. The solvent of claim 1 where the compound is at a concentration sufficient to maintain substrate activity for more than six months in solution at 4° C.
 3. The solvent of claim 1 being a microemulsion.
 4. The solvent of claim 1 as a ready-to-use non-toxic liquid.
 5. A composition for detecting the presence of lacZ gene comprising the solvent of claim 1 and an effective amount of chromogenic substrate selected from the group consisting of substituted indolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal), and combinations thereof.
 6. A composition for detecting the presence of lacZ gene comprising the solvent of claim 1, and an effective amount of substituted indolyl-β-D-galactoside or halo-substituted indolyl-β-D-galactoside chromogenic substrate selected from the group consisting of 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 6-chloro-3-indolyl-β-D-galactopyranoside n-methylindolyl-β-D-galactopyranoside, and 5-iodo-3-indolyl-β-D-galactopyranoside.
 7. The composition of claim 5 further comprising a lac operon inducer.
 8. A method for inducing a lac operon in a screening assay, the method comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce the lac operon.
 9. The method of claim 8 where the lac operon induced is selected from the group consisting of E. Coli, Bacillus subtilis, phage, yeast, or in situ tissues.
 10. The method of claim 8 where the essential oil is selected from the group consisting of Abies alba, Aniba roseodora, Cedrus atlantica, Citrus aurantifolia, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus paradisi, Citrus reticulata, Citrus sinensis, Cupressus sempervirens, Juniperus communis, Juniperus virginiana, Picea mariana, Pinus pinaster, Pinus sylvestris, Ravensara aromatica, Rosmarinus officinalis, citrus extracts, pine terpenoids, conifers extracts, limonene oil and linseed oil.
 11. A method for detecting the presence of bacteria, the method comprising the step of contacting an agar plate with at least one essential oil in a concentration sufficient to induce detection of bacteria.
 12. The method of claim 11 where the essential oil is selected from the group consisting of Abies alba, Aniba roseodora, Cedrus atlantica, Citrus aurantifolia, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus paradisi, Citrus reticulata, Citrus sinensis, Cupressus sempervirens, Juniperus communis, Juniperus virginiana, Picea mariana, Pinus pinaster, Pinus sylvestris, Ravensara aromatica, Rosmarinus officinalis, citrus extracts, pine terpenoids, conifers extracts, limonene oil and linseed oil.
 13. A composition for detecting presence, activity, and/or expression of a lacZ gene, the composition comprising a chromogenic substrate to visibly indicate presence, activity, and/or expression of the lacZ gene, the substrate selected from the group consisting of substituted indolyl-β-D-galactoside or halo-substituted indolyl-β-D-galactoside; a solvent comprising at least one solubilizing agent selected from the group consisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), at least one essential oil, and combinations thereof; and at least one essential oil inducer.
 14. The composition of claim 13 where the inducer is not isopropylthio-β-D-galactoside (IPTG).
 15. The composition of 13 further comprising at least one antibiotic with the substrate.
 16. The composition of claim 15 where the antibiotic is selected from the group consisting of ampicillin, tetracycline, chloramphenicol, kanamycin, and combinations thereof.
 17. A self-contained pre-cast medium for detecting activity and/or expression or non-expression of a lacZ gene, the medium comprising an agar matrix and in or on the agar matrix a chromogenic substrate selected from the group consisting of substituted indolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside, and 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal); a solvent comprising at least one solubilizing agent selected from the group consisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), and at least one essential oil; and an inducer selected from the group consisting of isopropylthio-β-D-galactoside (IPTG) and an essential oil.
 18. The medium of claim 17 where the inducer is not isopropylthio-β-D-galactoside (IPTG).
 19. The medium of 17 further comprising at least one antibiotic with the substrate.
 20. The medium of claim 19 where the antibiotic is selected from the group consisting of ampicillin, tetracycline, chloramphenicol, kanamycin, and combinations thereof.
 21. The medium of claim 17 having functional stability for at least three months 4° C.
 22. The medium of claim 17 in a ready-to-use plate.
 23. A composition for detecting presence, activity, expression, and/or non-expression of a lacZ gene, the composition comprising a chromogenic substrate providing a visible indication of the presence, activity, expression, and/or non-expression of the lacZ gene, a non-toxic microemulsion solvent, and at least one inducer essential oil replacing isopropylthio-β-D-galactoside (IPTG).
 24. The composition of claim 23 as a ready-to-use liquid.
 25. A composition for detecting presence, activity, expression, and/or non-expression of a lacZ gene, the composition comprising a chromogenic substrate providing a visible indication of the presence of lacZ gene and/or lacZ gene activity; a first phase comprising at least one solubilizing agent selected from the group consisting of 1-methylpyrrolidone (NMP), N¹-dimethyl propylene urea (DMPU), propylene carbonate (PC), an essential oil, and combinations thereof; a second phase comprising sea pine turpentine in a concentration of at most 1% v/v of the composition, and; water; the composition being a microemulsion and having a flash point greater than 73° C.
 26. The composition of claim 25 where the flash point is 76° C.
 27. A composition comprising a chromogenic substrate selected from the group consisting of substituted indolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside, or 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal); a 1-methylpyrrolidone (NMP) solubilizing agent; and sea pine turpentine; the composition having a flash point greater than 73° C.
 28. The composition of claim 27 where the substituted indolyl-β-D-galactoside or halo-substituted indolyl-β-D-galactoside is selected from the group consisting of 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 6-chloro-3-indolyl-β-D-galactopyranoside n-methylindolyl-β-D-galactopyranoside, and 5-iodo-3-indolyl-β-D-galactopyranoside.
 29. The composition of claim 27 having about 1% sea pine turpentine.
 30. The composition of claim 27 having a flash point of 76° C.
 31. The composition of claim 27 having about 89% NMP.
 32. The composition of claim 27 being transport safe.
 33. The composition of claim 27 lacking methanol.
 34. A chromogenic substrate solution for selection of antibiotic resistant bacterial clones, the solution comprising a substrate selected from the group consisting of substituted indolyl-β-D-galactoside, halo-substituted indolyl-β-D-galactoside, or 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-gal), and at least one antibiotic selected from the group consisting of ampicillin, tetracycline, chloramphenicol, and combinations thereof. 